Project Summary/Abstract Endocytosis is a conserved pathway that internalizes macromolecules and transmembrane proteins and sorts them through the endo-lysosomal system to their destinations. This process is often dysregulated in many diseases including cancers, metabolic disorders and neurodegenerative diseases. Elucidating the endocytic mechanisms of transmembrane proteins will improve our understanding of biological processes, disease prognosis and therapeutic strategies. Our lab has identified the poorly known transmembrane protein, TMEM127, which we discovered to be mutated in hereditary neuroendocrine tumors, as a tumor suppressor, a negative regulator of mTOR signaling and a lysosomal membrane protein. However, the function and regulation of TMEM127 remain poorly defined. Our previous studies show that TMEM127 localizes to the plasma membrane and to endo-lysosomal vesicles and that its localization changes in response to nutrient conditions. Specifically, the lysosomal localization of TMEM127 increases upon amino acid stimulation. At the lysosome, TMEM127 interacts with the amino acid-sensitive, lysosomal-anchored, mTOR activating complex which leads to dampened mTOR signaling. How TMEM127 senses amino acids and whether TMEM127 translocation to the lysosome originates from the plasma membrane, or an endosomal compartment, is unknown. Recently, we observed that several C-terminally truncated TMEM127 mutants predominantly localize to the plasma membrane suggesting that an endocytic signal is lost after truncation. We also recently observed that TMEM127 co-localizes with clathrin, but not caveolin, two mediators of endocytosis, indicating that clathrin-mediated endocytosis (CME) may be involved. These observations implicate a novel endocytic mechanism involved in TMEM127 internalization and trafficking to the lysosome. The objective of this proposal is to define these endocytic mechanisms. In Aim 1, we will identify the endocytic motif responsible for TMEM127 internalization and trafficking. In Aim 2, we will determine if TMEM127 internalization occurs through CME. In Aim 3, we will determine if TMEM127 internalizes in response to amino acids. Successful completion of this proposal will provide novel insights into the endocytic mechanisms regulating TMEM127 and its response to amino acids, which could have broad implications for human health including a better understanding of cancers with TMEM127 mutations or increased mTOR signaling and disorders affecting metabolic and lysosomal homeostasis.